Lactobacillus plantarum and composition comprising same

ABSTRACT

A method for treating an immune disease caused by a Th1/Th2 imbalance in favor of a Th2 shift in a subject including administering a composition comprising  Lactobacillus plantarum  CJLP133 strain KCTC 11403BP to the subject in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 16/148,569filed on Oct. 1, 2018, which is a Continuation of U.S. application Ser.No. 14/832,214 filed on Aug. 21, 2015 (now U.S. Pat. No. 10,130,666issued on Nov. 20, 2018), which is a Continuation of U.S. applicationSer. No. 13/386,459 filed on Jan. 23, 2012, which is a Continuation ofPCT Application No. PCT/KR2009/004930 filed on Sep. 2, 2009, whichclaims priority to Korean Application No. KR 10-2009-0067015 filed inthe Republic of Korea on Jul. 22, 2009. The contents of all of which arehereby expressly incorporated herein by reference, in their entirety,into the present application.

This application includes an electronically submitted Sequence Listingin .txt format. The .txt file contains a Sequence Listing entitled“3881_0368PUS3_ST25.txt” created on Nov. 5, 2020 and is 2,667 bytes insize. The Sequence Listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The present invention relates to a novel Lactobacillus plantarum, andcompositions comprising the same. More particularly, the presentinvention relates to a novel Lactobacillus plantarum helpful for theprevention/treatment of an enteric disease and an immune disease and acomposition comprising the same.

BACKGROUND ART

Lactic acid bacteria which can be found in traditional fermented foodsuch as ‘Kimchi’ is in a symbiotic relationship with human within thedigestive tract, and digests fibers and composite protein. Thesemicroorganisms contribute to the digestive environment within human orother animals and named ‘probiotics’. These probiotics should have goodacid-resistance, bile acid-resistance and intestinal epithelialcell-adherence in order to be efficiently attached to the smallintestine when taken orally.

The Lactobacillus sp. is a representative bacteria that is found intraditional fermented food such as ‘Kimchi’. Lactobacillus sp. are rodshaped bacteria which are found within the gastrointestinal tract ofhuman or other animals, dairy products and vegetables, and causes homo-or hetero-fermentation. Lactobacillus sp. lowers the pH of thegastrointestinal tracts to depress the reproduction of harmful bacteriasuch as E. coli or Clostridium, and improves diarrhea or constipation.Also, these bacteria have a role for vitamin synthesis, anticarcinogenicactivities, lowering of cholesterol levels. Acidophillin which is aproduct of the lactic acid bacteria activity, also depresses the growthof Shigella, Salmonella, Staphylococcus, E. coli. Furthermore, lacticacid bacteria improves diarrhea by depressing the growth of the bacteriaresponsible for diarrhea and normalisation of intestinal flora. (Michaeland Phillipe, Probiotics and prebiotics: Effects on diarrhea, Thejournal of nutrition, Volume 137, March 2007, pages 803S-811S;Roberfroid, Prebiotics and probiotics: Are the functional foods?,American journal of clinical nutrition, Volume 71, June 2000, pages1682S-1687S).

At present there are increasing amount of research on developingprobiotics and feedstuff using previously mentioned characteristics ofthe Lactobacillus sp. Bacteria—caused diarhhea in livestocks leads tothe decrease of rate of gain and increase of mortality rate. Therefore,in order to prevent this occurring, adding antibiotics in the feeds havebeen generally accepted. However, the use of antibiotics in feeds isbeing further regulated and organic livestock husbandry is recommendeddue to problems occurring from antibiotic resistant-bacteria and theantibiotic-remains within the animals. (Korean patent Laid-Openpublication 1998-78358) (McEwen and Fedorka-Cray, Antimicrobial use andresistance in animals, Clinical infectious Disease, Volume 34, June2002, pages S93-S106).

Furthermore, Lactobacillus sp. is known to be effective in increasingthe immune response. Recently, immune diseases such as allergy or atopicdisease are increasing globally including Korea. Currently in Europe,bacteriotherapy aiming to cure these diseases by orally administeringlactic acid bacteria is ongoing. Research describes that Lactobacillusrhamnosus had decreased the incidence rate of atopic disease in children(Kalliomaki et al., Probiotics in primary prevention of atopic disease:a randomised placebo-controlled trial, Lancet, Volume 357, April 2001,pages 1076-1079). Also, it has been reported that the area and degree ofeczema had decreased in children with progressed atopy when they weretreated with Lactobacillus rhamnosus and Lactobacillus reuteri(Rosenfeldt et al., Effect of probiotic Lactobacillus strains inchildren with atopic dermatitis, Dermatologic and ocular diseases,Volume 111, February 2003, pages 389-395).

Although the exact mechanism of the increased immune effect of thelactic acid bacteria has not been revealed, the research aiming tounderstand the cause is currently ongoing and so far it is understoodthat the orally administered lactic acid bacteria inhabits within thegastrointestinal tract and influence the immune system. For example, ithas been reported that lactic acid bacteria from yogurt increases theactivity of Peyer's patch lymphocytes, and stimulates IgA response shownfrom experiments with both animals and human. In addition, lactic acidbacteria affects both innate and adaptive immune system. These bacteriakills the pathogenic bacteria in the gastrointestinal tract (innateimmunity), and also activates the macrophages which destroy and presentthe antigen to the T lymphocyte (adaptive immunity) producing variouscytokines and interleukins such as IL-12 and IL-18. The increasedsecretion of cytokines is a result of the activation of NF-κB and STATsignalling pathway in the macrophages stimulated by the cell wallcomponents of the lactic acid bacteria. Moreover, lactic acid bacteriaare professional antigen presenting cells and activate dendritic cellsin the lymph nodes and gastrointestinal mucous membranes resulting insecretion of increased levels of IL-12, IL-18 and TNFα. Furthermore,these bacteria also increase the membrane proteins of the dendriticcells which activates MEW class II and B7-2 which stimulates Tlymphocytes (Cross et al., Anti-allergy properties of fermented foods:an important immunoregulatory mechanism of lactic acid bacteria?,International Immunopharmacology, Volume 1, May 2001, pages 891-901).

T lymphocytes are essential in adaptive immune immunity and it iscomprised of the cell-mediated Th1 and antibody-mediated Th2 responses.During the Th1 response, production of cytokines from antigen presentingcells such as IL-2, IL-18, and Interferon (IFN) are dominant. But duringthe Th2 response, PGE2, IL-4 and IL-10 are dominant. The balance betweenthese two responses is important and various immune diseases may occurwhen the balance is interrupted. Th1 cells are mostly involved withinfection where Th2 cells are associated with allergic and inflammatoryresponses. In the case which Th2 cells are over activated, theproduction of IgE antibodies increases, and may cause allergic responsesto some proteins (pollen, food) which were not as harmful before.Therefore, it is important that Th1 and Th2 responses remain balanced asinstability may cause disease. In addition, it has been reported thatthe secretion of cortisol occurring from continuous stress may causecancer, atopy, allerty and autoimmune diseases, as in this case Th1response decreases and Th2 response increases (Elenkov and Chrousos,Stress hormones, Th1/Th2 patterns, pro/anti-inflammatory cytokines andsusceptibility to disease, Trends in Endocrinology and Metabolism,Volume 10, November 1999, pages 359-368).

It has been reported from an in vivo experiment that lactic acidbacteria increases the secretion of IFN-γ which is a Th1 cell cytokine,but depresses the secretion of IL-4 and IL-5 which are Th2 cellcytokines (Matsuzaki et al., The effect of oral feeding of Lactobacilluscasei strain Shirota on immunoglobulin E production in mice, Journal ofDairy Science, Volume 81, January 1998, pages 48-53). Also, anotherexperiment described that administration of lactic acid bacteria intoovalbumin-primed mice which shows a biased Th2 response resulted inincreased level of IFN-γ but decreased levels of IL-4, IL-5 and IgE.Furthermore, co-culture of spleen cells collected from these animalswith lactic acid bacteria caused the same pattern of cytokine productionas the in vivo experiment. However, the co-culture of T lymphocytes withlactic acid bacteria did not show the increase of IFN-γ suggesting thatantigen presenting cells such as macrophages or dendritic cells may benecessary for the production of IFN-γ from T lymphocytes (Kato et al.,Lactic acid bacterium potently induces the production of interleukin-12and interferon-gamma by mouse splenocytes, International Journal ofImmunopharmacology, Volume 21, February 1999, pages 121-131).Furthermore, it has been reported that the secretion of IL-12 and IL-18which are cytokines produced from macrophages or dendritic cellsincreased dose-dependently, when these cells were co cultured withlactic acid bacteria. Therefore, lactic acid bacteria balance theTh1/Th2 responses where Th2 response is dominant, by stimulating the Th1response and increasing IL-12 and IL-18 production (Cross et al.,Anti-allergy properties of fermented foods: an importantimmunoregulatory mechanism of lactic acid bacteria?, InternationalImmunopharmacology, Volume 1, May 2001, pages 891-901). Thus, lacticacid bacteria is beneficial for preventing or treating cancer, atopy,allergy and autoimmune diseases which is caused by the unbalance ofTh1/Th2 responses and Th2 dominant responses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph illustrating the acid-resistance of Lactobacillusplantarum CJLP133.

FIG. 2 shows a graph illustrating the bile acid-resistance ofLactobacillus plantarum CJLP133.

FIG. 3 shows a graph illustrating the intestinal epithelialcell-adherence properties of Lactobacillus plantarum CJLP133.

FIG. 4 shows a graph illustrating the concentrations of IL-12 which is acytokine inducing Th1 response from a mouse splenocyte. This splenocytewas pre-treated with ovalbumin which induces Th2 response and was thenco cultured with Lactobacillus plantarum CJLP133 and also with otherbacteria for comparison of IL-12 measurement.

FIG. 5 shows a graph illustrating the concentrations of IL-4 which is acytokine inducing Th2 response from a mouse splenocyte. This splenocytewas pre-treated with ovalbumin which induces Th2 response and was thenco cultured with Lactobacillus plantarum CJLP133 and also with otherbacteria for comparison of IL-4 measurement.

FIG. 6 shows a graph illustrating the concentrations of IL-12 and IL-10using ELISA from macrophage cell line RAW264.7 treated withLactobacillus plantarum CJLP133 compared with other types of lactic acidbacteria.

FIG. 7 shows a graph illustrating the concentrations of IL-12 and IL-10using ELISA from dendritic cell line JAWS II treated with Lactobacillusplantarum CJLP133 compared with other types of lactic acid bacteria.

FIG. 8 shows a graph illustrating the mRNA concentrations of IL-12p40and IL-18 using RT-PCR from macrophage RAW264.7 treated withLactobacillus plantarum CJLP133 compared with other types of lactic acidbacteria.

FIG. 9 shows a graph illustrating the mRNA concentrations of IL-12p40and IL-18 using RT-PCR from dendritic cell JAWS II treated withLactobacillus plantarum CJLP133 compared with other types of lactic acidbacteria.

FIG. 10A shows the thickness of the skin removed from the NC/Nga mousewith atopic dermatitis which was treated with lactic acid bacteria.

FIG. 10B shows the light microscopic photo of lymphocytes accumulatedwithin the inflammatory lesion of the skin removed from the NC/Nga mousewith atopic dermatitis which was treated with lactic acid bacteria.

FIG. 11A shows a graph which illustrates the number of eosinophils fromthe skin removed from the NC/Nga mouse with atopic dermatitis which wastreated with lactic acid bacteria.

FIG. 11B shows the light microscopic photo of eosinophil infiltrationwithin the skin removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 12A shows a graph which illustrates the number of mast cells fromthe skin removed from the NC/Nga mouse with atopic dermatitis which wastreated with lactic acid bacteria.

FIG. 12B shows the light microscopic photo of mast cell infiltrationwithin the skin removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 13A shows a graph which illustrates the number of nerve fibers fromthe skin removed from the NC/Nga mouse with atopic dermatitis which wastreated with lactic acid bacteria.

FIG. 13B shows the light microscopic photo of nerve fiber-infiltrationwithin the skin removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 14A shows the light microscopic photo of axillary lymphacytic glandremoved from the NC/Nga mouse with atopic dermatitis which was treatedwith lactic acid bacteria.

FIG. 14B shows the light microscopic photo of spleen removed from theNC/Nga mouse with atopic dermatitis which was treated with lactic acidbacteria.

FIG. 15A shows a graph illustrating the total number of cells countedfrom the axillary lymphacytic gland removed from the NC/Nga mouse withatopic dermatitis which was treated with lactic acid bacteria.

FIG. 15B shows a graph illustrating the total number of cells countedfrom the spleen removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 16A shows a graph illustrating the total number of T cells countedfrom the axillary lymphacytic gland removed from the NC/Nga mouse withatopic dermatitis which was treated with lactic acid bacteria.

FIG. 16B shows a graph illustrating the total number of T cells countedfrom the spleen removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 17A shows a graph illustrating the total number of B cells countedfrom the axillary lymphacytic gland removed from the NC/Nga mouse withatopic dermatitis which was treated with lactic acid bacteria.

FIG. 17B shows a graph illustrating the total number of B cells countedfrom the spleen removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 18A shows a graph illustrating IL-12 concentrations using ELISAfrom single cell suspension of axillary lymph node removed from theNC/Nga mouse with atopic dermatitis which was treated with lactic acidbacteria, after culturing with dust mite extract.

FIG. 18B shows a graph illustrating IL-12 concentrations using ELISAfrom single cell suspension of spleen removed from the NC/Nga mouse withatopic dermatitis which was treated with lactic acid bacteria, afterculturing with dust mite extract.

FIG. 19A shows a graph illustrating IFN-γ concentrations using ELISAfrom single cell suspension of axillary lymph node removed from theNC/Nga mouse with atopic dermatitis which was treated with lactic acidbacteria, after culturing with dust mite extract.

FIG. 19B shows a graph illustrating IFN-γ concentrations using ELISAfrom single cell suspension of spleen removed from the NC/Nga mouse withatopic dermatitis which was treated with lactic acid bacteria, afterculturing with dust mite extract.

The inventors separated and identified a novel strain of Lactobacillussp. from traditional fermented food in order to develop a bacteria moreefficient for controlling the imbalance of Th1/Th2 response caused fromexcessive Th2 response, than other known bacteria.

Therefore, the aim of the invention is to develop a novel strain ofLactobacillus sp. which has excellent acid-resistance, bileacid-resistance, intestinal epithelial cell-adherence and induceimproved immune response, especially by balancing the Th1/Th2 responsewhere excessive Th2 response occurs.

The other aim of the present invention is to provide a compositioncomposed of the mentioned novel strain of Lactobacillus sp. which isbeneficial for the prevention or the treatment of enteric diseases.

Another aim of the present invention is to provide a compositioncomposed of the mentioned novel strain of Lactobacillus sp. which isbeneficial for improving immune responses.

In order to achieve the mentioned aim, present invention providesLactobacillus plantarum CJLP133 (Deposited at the Korea ResearchInstitute of Bioscience and Biotechnology (KRIBB), located at 111Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea on Oct. 9,2008, Accession number: KCTC 11403BP).

Furthermore, present invention provides a composition composed ofLactobacillus plantarum CJLP133, which is beneficial for the preventionor the treatment of enteric diseases.

In addition, present invention provides a composition composed ofLactobacillus plantarum CJLP133, which is beneficial for the improvingimmune responses. The detailed description of the present inventionfollows below.

Lactobacillus plantarum CJLP133 is characterised as a novel strain ofLactobacillus plantarum which was separated and identified fromtraditional fermented food. The mentioned traditional fermented food arekimchi, fermented vegetables, soybean paste, soy sauce, fast-fermentedbean paste and salted fish, but not only limited to these.

The present Lactobacillus plantarum CJLP133 was 99.9% homologous withthe reference strain (Lactobacillus plantarum NBRC15891^(T), GenBankaccession number AB326351) showing highly molecular phylogeny confirmedby 16S rRNA sequencing. Therefore, mentioned microorganism has beenidentified as Lactobacillus plantarum, named as Lactobacillus plantarumCJLP133 and has been deposited at the Korea Research Institute ofBioscience and Biotechnology (KRIBB) on Oct. 9, 2008 (Deposit numberKCTC 11403BP). The 16S rRNA sequencing result of Lactobacillus plantarumCJLP133 is SEQ ID NO:1 in the Sequence Listing.

Lactobacillus plantarum CJLP133 in the present invention is a grampositive bacteria which is a facultative anaerobe, able to grow in bothaerotropic and anaerobic conditions. This bacteria is immotile,rod-shaped and does not form spores. The detailed morphological andphysiological characteristics of Lactobacillus plantarum CJLP133 arelisted in Table 1 according to the common criteria of this technicalfield.

TABLE 1 Morphological, physiological and biochemical characteristicsResults Morphology Rod Motility − Spore − Catalase − Homo-heterofermentation Facultative fermentation Proliferation at 15° C. +Proliferation at 45° C. − Proliferation at 3% NaCl + Anaerobicproliferation + CO₂ production with glucose − Sugar fermentationcharacteristics Glycerol − Erythritol − D-arabinose − L-arabinose −Ribose + D-xylose − L-xylose − Adonitol − Xyloside − Galactose +D-glucose + D-fructose + D-mannose + L-sorbose − Rhamnose − Dulcitol −Inositol − Mannitol + Sorbitol + D-mannoside − D-glucoside −Glucosamine + Amygdalin + Arbutin − Esculin + Salicin + Cellobiose +Maltose + Lactose + Melibiose + Saccharose + Trehalose + Innulin −Melizitose + D-raffinose + Amidon − Glycogen − Xylitol − Gentiobiose +D-turanose − D-lyxose − D-tagatose − D-fucose − L-fucose − D-arabitol −L-arabitol − Gluconate − 2 Gluconate − 5-Gluconate − +: Positivereaction −: Negative reaction

In order to store Lactobacillus plantarum CJLP133 for a long periodsafely, it is recommended to keep the bacteria in preservation liquidmixed with water and glycerol at −70° C. or in suspension in sterilised10% skimmed milk followed by lyophilisation.

Furthermore, Lactobacillus plantarum CJLP133 in the present invention isprobiotics, which is beneficial for intestinal cleansing and improvedimmune responses like other lactic acid bacteria.

In the present invention, ‘probiotics’ is a term understood as a livemicroorganism which is beneficial for improving the gastrointestinalenvironment within human and other animals, therefore contributing tothe host's health. Probiotics are live microorganisms with probioticactivity, and are beneficial for gut microbiota when delivered to humanor animals as mixed or single bacterial strain in a dried or fermentedform. In order to be an efficient probiotic microorganism, it isimportant that firstly these bacteria are able to pass through thestomach without the influence of gastric fluid and bile, so that thebacteria reaches and survives at the intestine to contribute to thehealth of gut microbiota. Therefore these bacteria must haveacid-resistance, bile acid-resistance, and intestinal epithelialcell-adherence. Secondly, these bacteria should be safe microorganisms,and safety assessments are performed using gelatine liquefaction test,phenylalanine deaminase test, ammonification test and hemolysis test.Lactobacillus plantarum CJLP133 in the present invention has excellentacid-resistance, bile acid-resistance and intestinal epithelialcell-adherence. Also, Lactobacillus plantarum CJLP133 showed a negativeresult for gelatine liquefaction test, phenylalanine deaminase test andammonification test, and showed α-hemolysis for hemolysis test provingthe safety.

Lactobacillus plantarum CJLP133 in the present invention is predicted tobe beneficial for intestinal health as it has excellent acid-resistance,bile acid-resistance and intestinal epithelial cell-adherence. Thus, inanother aspect, this invention provides a composition composed ofLactobacillus plantarum CJLP133 which is beneficial for the preventionor treatment of enteric diseases.

The mentioned composition composed of Lactobacillus plantarum CJLP133which is beneficial for the treatment or prevention of enteric diseasescan be used for mammals including human and preferably for livestockssuch as cows, horses, pigs. The mentioned ‘enteric diseases’ includeinfection of the gastrointestinal tract and inflammatory entericdiseases. For example, infectious diarrhea caused by pathogenicmicroorganisms (E. coli, Salmonella, Chlostridium), gastroenteritis,inflammatory enteric diseases, neurogenic colitis, overgrowth ofmicroorganism in small intestine, acute gastroenteritis are included butonly limited to these. The mentioned Lactobacillus plantarum CJLP133used for the present composition is preferred to be alive, although itcan be either used alive or killed. In general, live bacteria cures andimproves any symptoms caused by abnormal fermentation by gutmicroorganisms, and prevents harmful bacteria from adhering to theintestinal walls when administered in human or animals. Also, livebacteria produces lactate which works toward decreasing the intestinalpH, hence preventing the survival of the harmful bacteria. Furthermore,the administered live bacteria prevents the proliferation of harmfulbacteria and helps the activity of intestinal vili which absorbsnutrients by producing bacteriocin and peroxide. In addition, livebacteria is beneficial for producing material which helps the absorptionof nutrients and its use, improves the demand rate of feedstuff, andneutralise toxic compounds secreted from pathogenic bacteria.

The administration route of the present compound described in thisinvention is recommended to be through oral methods, although it is notlimited. The dose differs depending on the type of enteric disease, thedegree of symptoms, age, gender, race, purpose of administration(treatment or prevention), however, ten million to 100 billion bacteriacan be administered in adults in general.

Furthermore, Lactobacillus plantarum CJLP133 is not only beneficial forthe intestinal health but also accelerates immune responses noticeablycompared with other lactic acid bacteria. Lactobacillus plantarumCJLP133 increases the secretion of IL-12 inducing Th1 response in thespleen, and also suppresses the secretion of IL-4 which induces the Th2response. Also, Lactobacillus plantarum CJLP133 stimulates the antigenpresenting cells which regulate the T cell immune response such asmacrophage and dendritic cells. These antigen presenting cells thensecretes cytokines which induces the differentiation of Th1 cell fromthe cells so that the imbalance of Th1/Th2 is compensated, thereforeproving the fact that Lactobacillus plantarum CJLP133 has the abilityfor immune regulation. More detailed description of the increased immuneresponse caused by Lactobacillus plantarum CJLP133 is explained below.

Lactobacillus plantarum CJLP133 produced 7.3 to 9.5 times more IL-12which induces the Th1 response, and suppressed the production of IL-4which induces Th2 response by 3.2 to 12.1% compared with negativecontrols. The cytokines were measured from the splenocyte of a mousetreated with ovalbumin so that Th2 response is dominant. The immuneregulatory effect of Lactobacillus plantarum CJLP133 is superior toother lactic acid bacteria such as Lactobacillus rhamnosus (KCTC 5033),Lactobacillus casei (KCTC3109), Lactobacillus sakei CJLS118 (KCTC13416).Therefore Lactobacillus plantarum CJLP133 has immune regulatoryproperties as it balances the imbalance of Th1/Th2 response bysuppressing the Th2 response and stimulating the Th1 response.

In addition, it has been confirmed that Lactobacillus plantarum CJLP133improves immune responses by stimulating macrophages, shown byexperiments coculturing Lactobacillus plantarum CJLP133 with macrophages(RAW264.7) and dendritic cells (JAWS II). When Lactobacillus plantarumCJLP133 was cocultured with macrophages (RAW264.7) and dendritic cells(JAWS II), the secretion of IL-12 and IL-18 which both induces thedifferentiation of Th1 cells were increased but the secretion of IL-10which inhibits the differentiation of Th1 cells was depressed comparedto IL-12. This experiment also proves that Lactobacillus plantarumCJLP133 CJLP133 has immune regulatory properties as it balances theimbalance of Th1/Th2 response by stimulating the Th1 response.

IL-4 is secreted from Th2 cells which is essential for cellular immunityand is an anti-inflammatory cytokine which suppresses the secretion ofIL-12 from Th1 cells. Recently reports have described that the amount ofTh2 cells secreting IL-4 and IL-5 were increased in the blood and skinlesions of patients with atopic dermatitis (Miraglia et al., Immunedysregulation in atopic dermatitis, Allergy and Asthma Proceedings,Volume 27, November-December 2006, pages 451-455). Therefore, dominationof Th2 response and imbalance of Th1/Th2 response may cause diseasessuch as atopic dermatitis. Also, as it has been described earlier, theimbalance of Th1/Th2 response may cause illness. Diseases such ascancer, atopic dermatitis, allergy and autoimmune disease may occur whenTh1 response is decreased and Th2 response is increased (Elenkov andChrousos, Stress hormones, Th1/Th2 patterns, pro/anti-inflammatorycytokines and susceptibility to disease, Trends in Endocrinology andMetabolism, Volume 10, November 1999, pages 359-368). Hence,Lactobacillus plantarum CJLP133 may be used for the treatment of notonly atopic dermatitis, allergy by its immune regulatory propertiesbalancing Th1/Th2 responses, but also for the treatment of cancer orautoimmune diseases.

Therefore, in another aspect, present invention provides a compositioncomposed of Lactobacillus plantarum CJLP133, which is beneficial forincreasing immune responses. This composition is effective forincreasing immune responses due to the activity of Lactobacillusplantarum CJLP133 within. Also, this composition is effective for theprevention or treatment of diseases caused by the imbalance of Th1/Th2response and dominant Th2 response, due to the activity of Lactobacillusplantarum CJLP133 within. Hence, the present composition composed ofLactobacillus plantarum CJLP133 can be used for the prevention ortreatment of atopic dermatitis, allergy, cancer and autoimmune diseases.Autoimmune diseases such as asthma and hay fever could be prevented ortreated but not only limited to these.

The administration route of the present composition composed ofLactobacillus plantarum CJLP133 is recommended to be through oralmethods, but not limited to this. The dose differs depending on the typeof enteric disease, the degree of symptoms, age, gender, race, purposeof administration (treatment or prevention), however, ten million to 100billion bacteria can be administered in adults in general.

The described composition composed of Lactobacillus plantarum CJLP133which is beneficial for prevention or treatment of enteric diseases andincreasing immune responses, is free from side effects for use asmedicine, health functional food, cosmetics, feedstuff, and feedadditives, as it includes lactic acid with proven safety.

In the case that the described composition composed of Lactobacillusplantarum CJLP133 is used as medical substance, the composition can bemanufactured into conventional pharmaceutically acceptable carriers.This composition can be manufactured into an oral dosage formpreferably. For example, liquid form, suspension concentrate, powderform, granule form, tablet, capsule, pills or extract forms could beused for administration.

In formulating into a respective dosage form, pharmaceuticallyacceptable and required carriers or additives may be added inmanufacturing the dosage form. For example, at least one carrierselected from diluents, slip agents, binding agents, disintegratingagents, sweetening agents, stabilizers and preservative agents; and atleast one additive selected from flavouring agents, vitamins andantioxidants may be used in formulating the oral dosage forms.

Any pharmaceutically acceptable carriers and additives may be used.Specifically, it may be preferable that lactose, corn starch, soybeanoil, microcrystalline cellulose or mannitol is used as diluents;magnesium stearate or talc is used as slip agents; polyvinylpyrrolidoneor hydroxypropylcellulose is used as binding agents. Further, it may bepreferable that carboxymethylcellulose calcium, sodium starch glycolate,polacrilin potassium or crospovidone is used as disintegrating agents;white sugar, fructose, sorbitol or aspartame is used as sweeting agents;sodium carboxymethylcellulose, β-cyclodextrin, white wax or xanthan gumis used as stabilizers; and methyl p-hydroxybenzoate, propylp-hydroxybenzoate or potassium solvate is used as preservative agents.

Further, in addition to the above ingredients, natural herbs withMae-sil (Japanese apricot) flavor, lemon flavor, pineapple flavor orherb flavor, natural fruit juice, natural pigments such as chlorophyllinor flavonoid, sweeting ingredients such as fructose, honey, sugaralcohol or sugar, acidifiers such as citric acid or sodium citrate maybe used after mixing for a purpose of raising appetite.

Formulating methods and carriers and additives necessary for suchformulating are detailed in Remington's Pharmaceutical Sciences (19thed., 1995).

Furthermore, the present composition composed of Lactobacillus plantarumCJLP133 can be used as food. The food composition covers conventionaldaily-consumed general foods as well as health foods. If the foodcomposition is used in the health foods, it may be formulated intoconventional health food dosage forms known to the art, withsitologically acceptable carriers or additives. The health foods may beformulated, for example, into powder, granule, tablet, capsule,suspension, emulsion, syrup, liquid, extract, jelly or drink form. Assitologically acceptable carriers or additives, an arbitrary carrier oradditive usable in any forms to prepare may be used.

The present composition can also be used as cosmetics as it isbeneficial for the prevention and treatment of atopic dermatitis. Thecosmetic composition according to the present invention may beformulated into conventional form known to the cosmetic industries. Anycarrier or additive which is acceptable and necessary in manufacturing aspecific cosmetic form may be added.

The present composition can also be used as feed additives or feedstuff.

Used in the feed additives, the composition may be manufactured into aform of 20 to 90% highly concentrated liquid, powders or granules. Thefeed additive may additionally include at least one selected fromorganic acids such as citric acid, humalic acid, adipic acid, lacticacid and malic acid; phosphates such as sodium phosphate, potassiumphosphate, acidic pyrophosphates and polyphosphates (condensedphosphate); and natural antioxidants such as polyphenols, catechins,alpha-tocopherols, rosemary extracts, vitamin C, green tea extracts,licorice extracts, chitosan, tannic acids and phytic acids. Used in thefeed composition, the composition may be manufactured into aconventional animal feed form and include conventional feed ingredients.

The feed additives and the animal feed may additionally include crops,for example, crushed or shredded wheat, oats, barley, corn and rice;vegetable protein feeds, for example, feeds mainly consisting of rape,soybean and sunflower; animal protein feeds, for example, blood meal,meat meal, bone meal and fish meal; and sugar and dairy products, forexample, various dry ingredients consisting of milk powder and wheypowder, and may further include nutritional supplements, digestion- andabsorption-enhancers and growth promoters.

The feed additives may be administered to animals individually or incombination with other additives selected from edible carriers. Further,the feed additives may be topdressing, may be directly mixed with animalfeeds or may be easily administered to animals as oral dosage formsseparately from animal feeds. In case of being administered separatelyfrom animal feeds, the feed additives may be combined withpharmaceutically acceptable edible carriers and prepared intoimmediate-release formulations or sustained-release formulations, aswell known in the art. The edible carriers may be solid or liquid, forexample, corn starch, lactose, sucrose, soy flake, peanut oil, oliveoil, sesame oil and propylene glycol. In case solid carriers are used,the feed additives may be in a form of tablet, capsule, powder, trocheor lozenge, or may be a not-dispersed topdressing. If liquid carriersare used, the feed additives may have a form of soft gelatin capsules,syrup, suspension, emulsion or solution.

The feeds may include an arbitrary protein-containing organic grainflour which has been conventionally used to meet animals' appetite. Theprotein-containing grain flour typically consists of corn or soybeanflour or is a mix of corn/soybean flour.

In addition, the feed additives and the animal feeds may containadjuvants such as preservatives, stabilizers, wetting agents,emulsifiers and liquefying agent. The feed additives may be added toanimal feeds by means of dipping, spraying or mixing for use.

The animal feeds or feed additives according to the present inventionmay be applied to a diet for various animals such as mammals, poultryand fish. The mammals may be pets (for example, dogs, cats) as well aspigs, cows, sheep, goats and laboratory rodents; poultry such aschickens, turkeys, ducks, geese, pheasant and quail; and fish suchtrout, without limitation thereto.

As described above, Lactobacillus plantarum CJLP133 according to thepresent invention is a probiotics which is characterised byacid-resistance, bile acid-resistance, intestinal epithelialcell-adherence. This bacteria is also beneficial for the intestinalhealth and balances the imbalance of Th1/Th2 response caused byexcessive Th2 response by stimulating Th1 response. Therefore, the novelLactobacillus plantarum CJLP133 according to the present invention canbe used in a composition for treatment of enteric diseases and forincreasing the immune response, and for treatment or prevention ofdiseases caused by imbalance of Th1/Th2 response.

Hereinafter, the present invention will be described by the followingexamples in more detail. However, the purpose of these examples is onlyto illustrate the present invention, not to limit the scope of theinvention thereto in any way.

EXAMPLE 1: ISOLATION AND IDENTIFICATION OF MICROORGANISM LACTOBACILLUSPLANTARUM CJLP133 BACTERIA

Lactic acid bacteria Lactobacillus plantarum CJLP133 were streaked ontosolid MRS medium (Difco, USA) containing 1.5% agar and incubated at 30°C. for 24 hrs. Colonies confirmed as being purely separated were takenby a loop and incubated with MRS broth (Difco, USA) at 30° C. for 18 to24 hrs.

Then, the morphology and physiological properties of Lactobacillusplantarum CJLP133 bacteria were determined with API50CH and API50CHLkits (Bio-Me'reux) according to the methods disclosed in Kim et. al.,Leuconostoc inhae sp. nov., a lactic acid bacterium isolated fromkimchi, International Journal of Systematic and EvolutionalMicrobiology, Volume 53, July 2003, pages 1123-1126. The resultantmorphology and physiological properties of Lactobacillus plantarumCJLP133 bacteria were summarized in the above table 1.

Further, a sequence of 16S rRNA gene was analyzed for identification andclassification of the lactic acid bacteria. The sequence of 16S rRNAgene was determined and analyzed according to a method disclosed in Kimet. al., Leuconostoc kimchii sp. nov., a new species from kimchi.International Journal of Systematic and Evolutional Microbiology, Volume50, September 2000, pages 1915-1919. The sequencing result of CJLP133 islisted in sequence list SEQ ID N01.

Since Lactobacillus plantarum CJLP133 according to the present inventionhas the highest homology (99.9%) with Lactobacillus plantarum NBRC15891^(T) reference bacteria (GenBank accession number AB326351),Lactobacillus plantarum CJLP133 according to the present invention wasidentified as Lactobacillus plantarum, named as Lactobacillus plantarumCJLP133 and deposited to Korea Research Institute of Bioscience andBiotechnology (KRIBB) Oct. 9, 2008 (Accession number: KCTC 11403BP).

EXAMPLE 2: EXPERIMENT INVESTIGATING ACID-RESISTANCE AND BILEACID-RESISTANCE OF LACTOBACILLUS PLANTARUM CJLP133 USING ARTIFICIALGASTRIC JUICE AND ARTIFICIAL BILE

The acid-resistance experiment was performed using the artificialgastric juice modified and made referring to an experiment fromKobayashi et al (Kobayashi et al., Studies on biological characteristicsof Lactobacillus: II. Tolerance of the multiple antibiotic resistancestrain, L. casei PSR3002, to artificial digestive fluids. Japan Journalof Microbiology, Volume 29, July 1974, pages 691-697). In detail, theartificial gastric juice was made by adjusting the pH of MRS liquidmedium to pH 2.5 using 1N HCl and adding 100 unit/ml of pepsin followedby sterilisation.

The isolated Lactobacillus plantarum CJLP133 as described in Example 1was cultured in

MRS liquid medium at 37° C. for 18 hours and then centrifuged forprecipitation. Then the precipitation was washed with sterilised 0.85%NaCl twice. Then 10⁷ cfu/ml of the bacterial suspension was inoculatedon the control medium and artificial gastric juice for further cultureat 37° C. The total number of live bacteria was estimated at 0 and 3hour-post inoculation after diluting bacteria by ten times in phosphatebuffer including KH₂, PO₄, Na₂HPO, L-cysteine, HCl, Tween 80.

The bile acid-resistance experiment was performed using the artificialbile modified and made referring to an experiment from Casey et al(Casey et al., Isolation and characterisation of anti-Salmonella lacticacid bacteria from the porcine gastrointestinal tract, Letters inApplied Microbiology, Volume 39, 2004, pages 431-438). Bacteria wascultured on the MRS liquid medium added with 0.3% bile of bull, and thetotal number of bacteria was counted after 0, 12 and 24 hourspost-inoculation of the lactic acid bacteria likewise to the acidresistance-experiment.

The described acid-resistance and bile acid-resistance were also testedon other representative lactic acid bacteria such as Lactobacillus casei(KCTC3109), Lactobacillus sakei CJLS118 (KCTC 13416) and Lactobacillusrhamnosus GG (KCTC 5033), for comparison.

The results are illustrated in FIG. 1 and FIG. 2 . FIG. 1 shows a graphillustrating the acid-resistance of Lactobacillus plantarum CJLP133.FIG. 2 shows a graph illustrating the bile acid-resistance ofLactobacillus plantarum CJLP133.

According to the results illustrated in FIG. 1 and FIG. 2 ,Lactobacillus plantarum CJLP133 showed more acid-resistance and bileacid-resistance compared with other lactic acid bacteria. This showsthat the novel bacteria described in this invention is capable ofreaching and surviving at the intestine without being influenced by thegastric juice or bile at the intestine.

EXAMPLE 3: EXPERIMENTS TESTING INTESTINAL EPITHELIAL-ADHERENCE OFLACTOBACILLUS PLANTARUM CJLP133

Animal cell line HT-29 was provided by the Korean Cell Line Bank (KCLB)in order to test intestinal epithelial-adherence, and the methods wereused described in the research of Kim et al and Hirano et al (Kim etal., Probiotic properties of Lactobacillus and Bifidobacterium strainsisolated from porcine gastrointestinal tract, Applied Microbiology andBiotechnology, Volume 74, April 2007, pages 1103-1111, Hirano et al.,The effect of Lactobacillus rhamnosus on enterohemorrhagic Escherichiacoli infection of human intestinal cells in vitro, Microbiology andImmunology, Volume 47, 2003, pages 405-109).

HT-29 was cultured in RPMI 1640 (Gibco, USA) medium added with heatdeactivated 10% Fetal Bovine Serum, 1% L-Glutamine, penicillin G (100IU/mL) and streptomycin (100 mg/mL) at 5% CO₂, 37° C. In order to testadherence and detachment, 1.0×10⁵ cell/ML of HT-29 cells were plated onwells of a 24 well plate. The medium were exchanged every other day forculture of these cells until there was a complete monolayer settled.Complete monolayers of HT-29 were washed 5 times with 25° C. of PBSbuffer and RPMI1640 medium without antibiotics was added.

1.0×10⁹ of Lactobacillus plantarum CJLP133 were suspended in RPMI andinoculated in each wells to be cultured for 2 hours at 5% CO₂, 37° C.After the culture, the wells were washed with PBS buffer three times bystirring the plate at 200 rpm for 3 minutes, in order to remove anydetaching lactic acid bacteria and to test adherence properties. Afterthe wash, 0.2% trypsin-EDTA was added to detach any cells from thewells. The number of bacteria which was streak plated on MRS-agar platewas counted using serial dilution method with peptone number after beingcultured at 37° C. for 24 hours.

Also, in order to test the partial adherence properties of the lacticacid bacteria, same amount of the lactic acid bacteria used in theexperiment above was placed on top of HT-29 cells which were cultured ona cover glass sterilised with 70% alcohol for a day placed on a petridish. The number of lactic acid bacteria which were adhered to HT-29cells was counted by looking under the light microscope after beingdried and stained by Gram staining. Comparison experiments wereperformed using Lactobacillus sakei CJLS118 (KCTC 13416) andLactobacillus rhamnosus GG (KCTC 5033).

The results are illustrated in FIG. 3 . FIG. 3 shows a graphillustrating the intestinal epithelial cell-adherence properties ofLactobacillus plantarum CJLP133. The results illustrated in FIG. 3 showsthat Lactobacillus plantarum CJLP133 has better intestinalepithelial-adherence measured after 24 hours than other probiotics suchas Lactobacillus rhamnosus GG (KCTC 5033) and Lactobacillus sakeiCJLS118 (KCTC 13416). Especially, the adherence properties ofLactobacillus plantarum CJLP133 was much better than Lactobacillus sakeiCJLS118 (KCTC 13416). These results suggest that the novel bacterialstrain indicated in this invention could improve the intestinal healthby adhering to intestinal epithelia.

EXAMPLE 4: SAFETY ASSESSMENT OF LACTOBACILLUS PLANTARUM CJLP133

Hemolysis test, gelatin liquefaction test, hazardous metabolite(ammonification) test and phenylalanine deaminase test were performedaccording to the safety assessment methods suggested by the standard ofKorea Biotechnology Industry Organization to assess safety of thebacteria isolated from the Example 1. The obtained result is summarizedin table 2.

TABLE 2 Safety assessment for Lactobacillus plantarum CJLP133 Testsgelatin phenylalanine Bacteria liquefaction deaminase hemolysisammonification CJLP133 negative negative α-hemolysis, negative safe

Based on the above result, Lactobacillus plantarum CJLP133 was foundnegative in the gelatin liquefaction test, hazardous metabolite(ammonification) test, phenylalanine deaminase test. Hemolysis testshowed α-hemolysis which is irrelevant with pathogenic bacteria.Accordingly, Lactobacillus plantarum CJLP133 was confirmed as being safefor administration to a human being.

EXAMPLE 5: EVALUATION OF STIMULATING PROPERTIES OF IL-12 PRODUCTIONAFTER TREATING MOUSE SPLENOCYTE

Lactobacillus plantarum CJLP133 was added to mouse splenocytes treatedwith ovalbumin biased towards Th2 response, in order to evaluate thestimulating properties of Lactobacillus plantarum CJLP133 inducing IL-12production which is a Th1 response inducing cytokine. For theexperiment, methods were referred from reports from Fujiwara et al.(Fujiwara et al., A double-blinded trial of Lactobacillus paracaseistrain KW3110 administration for immunomodulation in patients withpollen allergy, Allergology International, 2005, volume 54, pages143-149) and Fujiwara et al. (Fujiwara et al., The anti-allergic effectsof lactic acid bacteria are strain dependent and mediated by effects onboth Th1/Th2 cytokine expression and balance, International Archives ofAllergy and Immunology, 2004, Volume 135, pages 205-215).

5 of 6 weeks old female Balb/c mouse were immunised with a mixedsolution composed of 1.538 mL of 13 mg/mL alumhydroxide (Sigma), 10 mgovalbumin, 0.4615 mL of PBS. This solution was mixed well and kept atroom temperature for 20 minutes for reaction, and 0.2 mL (1 mg OVA+2 mgalum) was injected peritoneally into the mouse. The same amount ofsolution was injected into these mice on day 6 post injection, forboosting. Mice were sacrificed on day 13 post injection to remove thespleen. 100 μl (4×106) splenocytes taken from the spleen, 50 μl ofkilled bacteria for testing and 50 μl (4 mg/ml) of ovalbumin were addedand placed onto cell culture well plate in DMEM-10 medium for 7 days at10% CO₂ for culture. After the 7 days culture, the supernatant fluid wastaken for measuring IL-12 concentrations using IL-12 ELISA kit(Biosource).

The killed bacteria for testing described above were obtained as writtenbelow.

The test bacteria was inoculated in MRS liquid medium (Difco) andcultured for 24 hours at 37° C. Then the culture medium was centrifugedat 13000 rpm for 1 minute followed by 2 times of washing usingphysiological saline, and the bacteria was obtained. The obtainedbacteria was heated at 100° C. for 10 minutes suspended in steriliseddistilled water (same amount as the original culture medium). Then thesuspension was centrifuged at 13000 rpm for 1 minute and the bacteriawas resuspended in DMEM medium at the concentration of 50 μg/ml and 5μg/ml. Test bacteria was Lactobacillus plantarum CJLP133, and the sameexperiment was performed using Lactobacillus rhamnosus GG (KCTC 5033),Lactobacillus casei (KCTC 3109) and Lactobacillus sakei CJLS118 (KCTC13416) for comparison.

The mentioned IL-12 assay was performed by using IL-12 ELISA kit andprovided instructions. The O.D value of the control sample providedwithin the kit was measured and referring to the equation, the amount ofIL-12 from the samples was calculated. The results are illustrated inFIG. 4 .

FIG. 4 shows a graph illustrating the concentrations of IL-12 which is acytokine inducing Th1 response from a mouse splenocyte. This splenocytewas pre-treated with ovalbumin which induces Th2 response and was thenco cultured with Lactobacillus plantarum CJLP133 and also with otherbacteria for comparison of IL-12 measurement.

According to the result shown in FIG. 4 , Lactobacillus plantarumCJLP133 markedly induces the production of IL-12 which is a Th1 responseinducing cytokine, compared to other bacteria. Therefore, it has beenproved that Lactobacillus plantarum CJLP133 in this inventionefficiently induces Th1 response in mouse with biased Th2 response.

EXAMPLE 6: EVALUATION OF SUPPRESSION OF IL-4 PRODUCTION AFTER TREATMENTWITH MOUSE SPLENOCYTE

In order to test whether Lactobacillus plantarum CJLP133 suppresses theproduction of IL-4 which is a Th2 response inducing cytokine,Lactobacillus plantarum CJLP133 was added to mouse splenocyte biasedwith Th2 response due to ovalbumin treatment. ELISA kit was used as hasbeen described in Example 5, but IL-4 kit (Biosource) was used insteadof IL-12 kit. Other experimental conditions were the same and theresults are shown in FIG. 5 .

FIG. 5 shows a graph illustrating the concentrations of IL-4 which is acytokine inducing Th2 response from a mouse splenocyte. This splenocytewas pre-treated with ovalbumin which induces Th2 response and was thenco cultured with Lactobacillus plantarum CJLP133 and also with otherbacteria for comparison of IL-4 measurement.

FIG. 5 shows that Lactobacillus plantarum CJLP133 suppresses theproduction of IL-4, a cytokine that induces Th2 response, so that itsuppresses Th2 response in mouse splenocytes biased towards Th2response.

EXAMPLE 7: EXPERIMENTS TESTING THE EXPRESSION OF CYTOKINES IL-12P40 ANDIL-18 WHICH INDUCES THE DIFFERENTIATION INTO TH1 LYMPHOCYTE, ANDEXPRESSION OF CYTOKINE IL-10 WHICH SUPPRESSES THE DIFFERENTIATION INTOTH1 LYMPHOCYTES

Antigen presenting cells such as macrophages and dendritic cells produceIL-12 and IL-18 which induces the differentiation of Th1 cells from Th0cells, and on the other hand produce IL-10 which suppresses thedifferentiation of Th1 cells from Th0 cells. Further experiments wereperformed in order to investigate the effect of lactic acid bacteria onthe production of IL-12, IL-10 and IL-18 by macrophages and dendriticcells.

5×10^(7/) mL of test bacteria was added to macrophage cell line RAW264.7and cultured for 48 hours at 37° C., 10% CO₂. Then the medium was takento measure the concentrations of IL-12p40 and IL-10 using ELISA method.Also the bacteria was added to dendritic cell line JAWS II using thesame method as above, and the concentrations of IL-12p40 and IL-10 weremeasured using ELISA.

The test bacteria was Lactobacillus plantarum CJLP133, andlipopolysaccharide was used as a positive control. Lactobacillusrhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109) andLactobacillus sakei CJLS118 (KCTC 13416) were also used in theexperiment to compare the results.

Measurement of concentrations of cytokines was performed using ELISAmethod. IL-12p40 kit (BD BioSciences, USA) and IL-10 kit (BDBioSciences, USA) was used for measurement of IL-12 and IL-10respectively. The results are illustrated in FIG. 6 and FIG. 7 .

FIG. 6 shows a graph illustrating the concentrations of IL-12 and IL-10using ELISA from macrophage cell line RAW264.7 treated withLactobacillus plantarum CJLP133 compared with other types of lactic acidbacteria.

FIG. 7 shows a graph illustrating the concentrations of IL-12 and IL-10using ELISA from dendritic cell line JAWS II treated with Lactobacillusplantarum CJLP133 compared with other types of lactic acid bacteria.

According to the results illustrated in FIG. 6 and FIG. 7 ,Lactobacillus plantarum CJLP133 produces IL-12 which is a cytokine thatinduces the differentiation into Th1, and produces less IL-10 which is acytokine that suppresses the differentiation into Th1, compared withIL-12. Also Lactobacillus plantarum CJLP133 produces a more noticeableamount of IL-12 than other lactic acid bacteria.

Furthermore, in order to investigate the amount of IL-12 and IL-18 at agenetic level, 5×10^(7/) mL of test bacteria was added to macrophagecell line RAW264.7 for culture at 37° C., 10% CO₂ for 6 hours. Then thetotal RNA was extracted and the mRNA concentration of IL-12 and IL-18was measured using RT-PCR. The test bacteria was also inoculated andcultured with dendritic cell line JAWS II in order to measure the mRNAamount of IL-12 and IL-18 using RT-PCR.

The results are illustrated in FIG. 8 and FIG. 9 .

FIG. 8 shows a graph illustrating the mRNA concentrations of IL-12p40and IL-18 using RT-PCR from macrophage RAW264.7 treated withLactobacillus plantarum CJLP133 compared with other types of lactic acidbacteria.

FIG. 9 shows a graph illustrating the mRNA concentrations of IL-12p40and IL-18 using RT-PCR from dendritic cell JAWS II treated withLactobacillus plantarum CJLP133 compared with other types of lactic acidbacteria.

According to the results illustrated in FIG. 8 and FIG. 9 ,Lactobacillus plantarum CJLP133 stimulates the production of mRNAinducing the formation of IL-12 and IL-18 which are cytokines thatinduce the differentiation into Th1 cells. Particularly, Lactobacillusplantarum CJLP133 produces more noticeable amount of IL-12 mRNA comparedwith other lactic acid bacteria.

EXAMPLE 8: IN VIVO EXPERIMENT OF THE EFFECT OF LACTOBACILLUS PLANTARUMCJLP133 STRAIN ON ATOPIC DERMATITIS

Experimental Animal Breeding and Grouping

Lactic acid bacterial strain was administered orally into NC/Nga mousewhich were caged for a week after arriving at the animal unit as 4 weeksold. The temperature was kept at 24±2° C., and the light cycle was 12hours. Feedstuff was in powder form without any antibiotics added.Lactic acid bacteria was administered orally into mice by mixing withfeedstuff evenly for 10 weeks (1×10¹⁰ cfu/animal). Atopic dermatitis wasinduced in animals 6 weeks post administration of lactic acid bacteria,by applying Biostir AD ointment (Biostir, Japan) for 5 weeks. Mice weregrouped as non induction group without induction of atopic dermatitis,control group induced with atopic dermatitis, a group with atopicdermatitis administered with lactic acid bacteria. 8 mice were used pereach group (Table 3). Lactobacillus sakei CJLS118 (KCTC13416),Lactobacillus rhamnosus GG (KCTC 5033) were used as test lactic acidbacteria. Also, CJLP55 (KCTC11401BP), CJLP56 (KCTC 11402BP) and CJLP136(KCTC 11404BP) which were developed by the applicant of the presentinvention were used. Lastly, CJLP133 (KCTC 11403BP) from the presentinvention was used as well.

TABLE 3 Induction Administered lactic acid of atopic Group bacteriadermatitis non-induction X Control group ◯ GG GG (KCTC 5033) ◯ LP55CJLP55 (KCTC 11401BP) ◯ LP56 CJLP56 (KCTC 11402BP) ◯ LP133 CJLP133 (KCTC11403BP) ◯ LP136 CJLP136 (KCTC 11404BP) ◯ LS118 CJLS118 (KCTC 13416) ◯LA12 CJLA12 ◯

Induction of Atopic Dermatitis

Fur was removed from the back upto the back of the ear of NC/Nga mouseusing a depilater and any remaining fur was removed using a depilatorycream. 4% SDS solution was sprayed on to the application area to removethe lipid component and dried for an hour. A flat stick was used toapply 100 mg of Biostir AD ointment (Biostir, Japan) at the back and theearflap evenly. The Biostir AD ointment was applied 10 times in total byapplying twice per week for 5 weeks.

Tissue Staining

Atopic dermatitis is characterised by thickening of the skin,penetration of immune cells such as lymphocytes, monocytes, eosinophils,mast cells into the tissue causing inflammation. Also, the nerve fiberextends abnormally to the epidermis causing itchiness. Therefore, theskin of the mouse with atopic dermatitis was removed to examine andcount the numbers of the immune cells and nerve fibers mentioned above.

Five weeks after inducing atopic dermatitis, the mouse was culled forremoval of the skin. The skin was fixed with Accustain formali-freefixative solution and was paraffin blocked. The block was cut by 5 μm,and went under hematoxylin/eosin staining to check the thickness of theskin (epidermis+derma). The tissue was also used to investigate theaccumulation of lymphocytes within the inflammatory lesion using thelight microscope of the 2×2 mm area. Also, the tissue was stained withToluidine blue to detect mast cells, and Congo red to detect eosinophilsin the 2×2 mm area using light microscope. Mast cells and eosinophilswere counted by looking at the area from the epidermis to the muscletissue. Immunohistochemistry was used in order to detect the penetrationof nerve fibers into the skin tissue. Anti-protein gene product (PGP.5)antibody was used for detection, and biotin-conjugated goat anti-rabbitantibody and peroxidise-conjugated streptavidin was added sequentiallyfor colour formation by peroxidise reaction.

The results are illustrated in FIGS. 10A to 13B.

FIG. 10A shows the thickness of the skin removed from the NC/Nga mousewith atopic dermatitis which was treated with lactic acid bacteria.

FIG. 10B shows the light microscopic photo of lymphocytes accumulatedwithin the inflammatory lesion of the skin removed from the NC/Nga mousewith atopic dermatitis which was treated with lactic acid bacteria.

FIG. 11A shows a graph which illustrates the number of eosinophils fromthe skin removed from the NC/Nga mouse with atopic dermatitis which wastreated with lactic acid bacteria.

FIG. 11B shows the light microscopic photo of eosinophil infiltrationwithin the skin removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 12A shows a graph which illustrates the number of mast cells fromthe skin removed from the NC/Nga mouse with atopic dermatitis which wastreated with lactic acid bacteria.

FIG. 12B shows the light microscopic photo of mast cell infiltrationwithin the skin removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

FIG. 13A shows a graph which illustrates the number of nerve fibers fromthe skin removed from the NC/Nga mouse with atopic dermatitis which wastreated with lactic acid bacteria.

FIG. 13B shows the light microscopic photo of nerve fiber-infiltrationwithin the skin removed from the NC/Nga mouse with atopic dermatitiswhich was treated with lactic acid bacteria.

According to the results described above, the thickness of the skin ineach experimental group composed of the NC/Nga mice induced with atopicdermatitis was approximately 100 μm.

However, the thickness of the skin in mice administered with CJLP133 wasapproximately 50 μm which was almost halved compared to others (FIG. 10a ). Also, during observation of the penetration of lymphocytes andmonocytes, it was found that markedly less numbers of immune cells werepresent in the CJLP133 administered group, whereas more immune cellswere stained purple in the control and GG group (FIG. 10 b ).

Investigation of eosinophils and mast cells within the inflammatorylesion showed that the group induced with atopic dermatitis had largernumbers of eosinophils and mast cells compared with the non-inductiongroup. However, the group that received CJLP133 had markedly lesseosinophils and mast cells compared with control group and groupsreceiving other lactic acid bacteria (FIG. 11 a and FIG. 12 a ). Lightmicroscopic photos showed that there were more blue eosinophils and mastcells in control group and GG administered group. However, it also showsthat there were less eosinophils and mast cells in the CJLP133administered group (FIG. 11 b and FIG. 12 b ).

Immunohistochemical analysis showed that the penetration of nerve fiberswas not observed in the non-induction group. However, in the controlgroup, many brown nerve fibers were found (FIG. 13 a ). In the groupsadministered with lactic acid bacteria, the number of nerve fiberspenetrating decreased, and especially marked numbers of nerve fiberswere decreased in CJLP55, CJLP133, CJLP136 administered groups (FIG. 13b ).

Investigation of the composition of axillary lymph node and splenocyteAxillary lymph node (ALN) is an important immune organ which plays amajor role in the animal model of chronic atopic dermatitis. There arereports from some patients with serious chronic atopic dermatitis, thatthe size of their axillary lymph node was increased. In the NC/Nga mousewhich is an animal model of atopic dermatitis induced by dust mites,axillary lymph node has been the target lymph node for investigation inmany researches. Therefore, in the present study, axillary lymph nodeand spleen which is the main immune organ was removed in order toobserve the change in size and composition of the cells.

Five weeks after the induction of atopic dermatitis, the mouse wasculled to remove the axillary lymph node and the spleen to compare thesize. Then red blood cells were removed from these organs to obtainsingle cell suspension. 1×10⁶ cells in suspension was distributed ineach FACS tubes and were stained with anti-Thy1.2-FITC, anti-CD19-FITC,anti-F4/80-FITC, anti-CD11c-FITC for FACS analysis in order to study thecomposition of T and B lymphocytes. The results are shown in FIG. 14 toFIG. 17 .

FIG. 14 shows the light microscopic photo of axillary lymph node (A) andspleen (B) removed from the NC/Nga mouse with atopic dermatitis whichwas treated with lactic acid bacteria.

FIG. 15 shows a graph illustrating the total number of cells countedfrom the axillary lymph node (A) and spleen (B) removed from the NC/Ngamouse with atopic dermatitis which was treated with lactic acidbacteria.

FIG. 16 shows a graph illustrating the total number of T cells countedfrom the axillary lymph node (A) and spleen (B) removed from the NC/Ngamouse with atopic dermatitis which was treated with lactic acidbacteria.

FIG. 17 shows a graph illustrating the total number of B cells countedfrom the axillary lymph node (A) and spleen (B) removed from the NC/Ngamouse with atopic dermatitis which was treated with lactic acidbacteria.

According to the results above, the size of axillary lymph node hadincreased in the control group induced with atopic dermatitis, and thesize was similar in GG administered group. However, the size of axillarylymph node of the group administered with CJLP55, CJLP56, CJLP133,CJLP136, CJLS118 was smaller than the control group (FIG. 14A). Thespleen did not show much difference in size comparing the groups (FIG.14B). The number of cells isolated from the axillary lymph node was 4.5times larger in control group compared with non-induction group.However, the number of cells was significantly smaller in the groupadministered with CJLP55, CJLP133, CJLP136 compared with the controlgroup (FIG. 15A). The number of cells in the spleen did not show muchdifference among the different groups (FIG. 15B).

Investigation of the T and B lymphocytes was performed in the axillarylymph node and spleen by staining and FACS analysis. The number of T andB lymphocytes from the axillary lymph node increased five times in thecontrol group induced with atopic dermatitis. However, the number ofcells in all the groups administered with CJLP55, CJLP56, CJLP133,CJLP136 was significantly decreased compared with control group.Especially, the number of cells was remarkably decreased in the CJLP133group compared with other lactic acid administered groups (FIG. 16A andFIG. 17A). The number of T and B lymphocytes did not show muchdifference (FIG. 16B and FIG. 17B).

5) Ability of cytokine production of axillary lymph node cells andsplenocytes IL-12 which is produced mainly by macrophages induces thedifferentiation of Th0 lymphocyte into Th1 lymphocyte. IFN-γ which isproduced by Th1 lymphocyte not only activates macrophages but alsosuppresses the differentiation into Th2 cells and its activity.Therefore, the changes in concentrations of IL-12 and IFN-γ producedwere measured.

The single cell suspension was obtained from the previously mentionedexperiment 4) Investigation of the composition of axillary lymph nodeand splenocyte. The suspension was added to each of the wells of the24-well plate in a concentration of 5×10⁶ cells per plate, and 10 μg/mlof dust mite (Dermatophagoides farinae body, Dfb) extraction was addedas well. The plate was kept at 37° C. for 48 hours for culture, and thenthe concentrations of IFN-γ and IL-12 was measured using ELISA. Theresults are illustrated in FIG. 18 and FIG. 19 .

FIG. 18 shows a graph illustrating IL-12 concentrations using ELISA fromsingle cell suspension of axillary lymph node (A) and spleen (B) removedfrom the NC/Nga mouse with atopic dermatitis which was treated withlactic acid bacteria, after culturing with dust mite extract.

FIG. 19 shows a graph illustrating IFN-γ concentrations using ELISA fromsingle cell suspension of axillary lymph node (A) and spleen (B) removedfrom the NC/Nga mouse with atopic dermatitis which was treated withlactic acid bacteria, after culturing with dust mite extract.

According to the results, the concentrations of IL-12 (FIG. 18 ) andIFN-γ (FIG. 19 ) were remarkably increased in the group receivingCJLP133 compared with control group. Also, the concentration of thesecytokines were remarkably larger compared with other groups administeredwith other known lactic acid bacteria.

All the groups administered with CJLP55, CJLP56, CJLP133, CJLP136 showedan improvement of atopic dermatitis symptoms, summarising the resultsfrom the experiment 1) to 5) as described above. Especially, CJLP133 waseven more effective for atopic dermatitis compared with other knownlactic bacteria, supported by its cellular and molecular effects on theanimals, such as the size of axillary lymph node, number of cells in theaxillary lymph node, penetration of immune cells or nerve cells intoinflammatory skin lesions, and the balance of Th1/Th2 cytokines.

EXAMPLE 9: MANUFACTURING PROBIOTICS INCLUDING LACTOBACILLUS PLANTARUMCJLP133

Lactobacillus plantarum CJLP133 identified as described in Example 1 wasmass produced and lyophilised to be manufactured into probiotics inorder to be used as medicine, food, feedstuff, feed additives, ormaterial for cosmetics.

For mass production, the bacteria was cultured in MRS liquid medium(Difco) added with 25% NaOH to reach pH 6.0 for 18 hours at 37° C. Thenthe bacteria was obtained by centrifugation. The bacteria was thenfrozen at −40° C. using 5% dextrin and 10% skimmed milk as a protector,and the dried bacteria was ground using a mixer to be in a power form.The powdered bacteria was stored and packed in an aluminium pouch bagmixed with an appropriate amount of diluting agent such as glucose,lactose, skimmed milk.

The manufactured probiotics can be used as feed probiotics by mixingwith feed material such as grain powder; as medicine or health food in aform of tablets or capsules mixed with carriers or additives; ascosmetics after being mixed with other cosmetic material. The probioticscould be used in various industries such as medicine, food, feedstuff,cosmetics according to the conventional methods in the art.

What is claimed is:
 1. A method for treating an immune disease caused bya T-helper 1/T-helper 2 (Th1/Th2) imbalance in favor of a Th2 shift in asubject, comprising: administering a composition comprisingLactobacillus plantarum CJLP133 strain deposited under Accession No.KCTC 11403BP to the subject in need thereof.
 2. A method for treating animmune disease in a subject, comprising: administering a compositioncomprising Lactobacillus plantarum CJLP133 strain deposited underAccession No. KCTC 11403BP to the subject in need thereof, wherein theimmune disease is caused by a T-helper 1/T-helper 2 (Th1/Th2) imbalanceselected from the group consisting of allergic diseases, atopy, cancer,and enteropathy.
 3. The method of claim 1, wherein the composition isselected from the group consisting of pharmaceuticals, food, animalfeed, feed additives and cosmetics.
 4. The method of claim 2, whereinthe composition is selected from the group consisting ofpharmaceuticals, food, animal feed, feed additives and cosmetics.
 5. Themethod of claim 1, wherein the subject is a human.
 6. The method ofclaim 2, wherein the subject is a human.
 7. The method of claim 1,wherein the composition is administered orally.
 8. The method of claim2, wherein the composition is administered orally.